JP2009538366A - Extraction of highly unsaturated lipids with liquid dimethyl ether - Google Patents

Abstract

  A method for obtaining a lipid containing a highly unsaturated fatty acid from a plant or animal material, the method comprising contacting the material with liquid dimethyl ether to obtain a lipid-containing dimethyl ether solution and a residue of the plant or animal material, the solution comprising: A method comprising the steps of separating from residues of plant or animal material and recovering lipids from the solution.

Description

  The present invention relates to a separation technique. In particular, the invention relates to liquid dimethyl ether (DME), and in some cases near-critical carbon dioxide, for example dry or partially dry plants or seeds (including marine or terrestrial species) or animals. Extract materials such as animal products (including marine or terrestrial species) and extract rich in highly unsaturated lipids, especially highly unsaturated complex lipids, and in some cases, nutraceuticals ( nutraceutical) or to obtain a residue that is useful for extraction of water-soluble enzymes and / or proteins.

Background Polyunsaturated lipids (lipids having 3 or more unsaturated sites and 18 or more carbons in a fatty acid chain) have various metabolic roles in the human body. They are important for neonatal brain and vision development and may also be advantageous for cardiovascular health, mental health, immune status and inflammatory status. The biological properties of these lipids usually depend on the type of fatty acid present, with lipids containing highly unsaturated fatty acids being the most bioactive. In general, these polyunsaturated fatty acids are found only in significant amounts in complex lipids in terrestrial plants and animals, but may also occur in both neutral and complex lipids in marine animals.

  Phospholipids are a subset of complex lipids. Phospholipids are an essential component of every mammalian cell membrane and play an important role in maintaining the fluidity of the cell membrane and the passage of molecules through the membrane. Polyunsaturated arachidonic acid (C20: 4.w-6) is absent or present at very low concentrations in animal-derived secondary products (eg, phospholipids from non-human milk). Arachidonic acid is extremely important for the development of newborns, so this fatty acid is supplemented to neonatal formulas made from non-human milk. There is therefore a need to ensure a source of this fatty acid for this purpose. The complex lipids of many animal tissues (especially organs and glands) are as rich in arachidonic acid as eggs.

  Moss and ferns are also known to contain high levels of arachidonic acid in complex lipid form. Therefore, it is particularly desirable to find an extraction technique that can recover this highly unsaturated fatty acid (HUFA) in complex lipid form, since the complex lipid form of the fatty acid provides protection against oxidation.

  Marine organisms (micro and macroalgae, fish bodies, eggs and liver, molluscs, invertebrates) are considered to be HUFAs eicosapentanoic acid (C20: 5.w-3) and docosa in neutral and / or complex lipid forms. A rich source of hexanoic acid (C20: 6.w-3). These fatty acids are also needed for supplementing neonatal formulas and for controlling neurological, cardiovascular, inflammation, and lipid content in the blood. It would also be desirable to find an extraction technique that can recover these polyunsaturated fatty acids.

  Similarly, seeds from certain plants, especially seeds from pinus and podocarp trees, are non-methylene interrupted polyunsaturated fatty acids (C20: 3 and C20: 4) Contain rich complex lipids. Non-methylene interrupted fatty acids are used for controlling satiety and as possible anti-inflammatory drugs. Therefore, there is a need to find an extraction technique that can recover these polyunsaturated fatty acids.

The extraction of neutral lipids using supercritical CO ₂ is well known, especially in the extraction of seed oil. The disadvantage of these methods is that they generally require large high pressure vessels (typically using pressures above 300 bar) to contain the raw materials, which makes the production plant very expensive. To do. Furthermore, since the oil has a very low solubility in supercritical CO ₂ (usually 1 g oil per 100 g solvent), high flow rates and long extraction times are also required. There are very few publications on the extraction of lipids from marine species. US Pat. No. 6,083,536 describes a method for extracting nonpolar lipids from crude lyophilized mussel powder to obtain a nonpolar lipid fraction useful for the treatment of inflammatory conditions. Fresh mussels are stabilized with tartaric acid prior to lyophilization and CO ₂ extraction. Composition data of the extract Izu described, since complex lipids are insoluble in CO _2, complex lipids are not extracted.

US Pat. No. 4,367,178 describes a method for purifying crude soybean lecithin using supercritical CO ₂ to extract neutral lipids, leaving insoluble phospholipids, thereby concentrating the phospholipids in lecithin. Crude lecithin is produced by conventional degumming of soybean oil. To overcome the limitations of CO _2, using cosolvents, such as ethanol to increase the dissolving power of the supercritical CO ₂ (solvent power) is proposed.

EP 1,044,245 A2 extracts dried eggs with supercritical CO ₂ first to remove neutral lipids and then with supercritical CO ₂ and an organic co-solvent (ethanol) that is liquid at room temperature, or Describes a method of extracting phospholipids either by extraction with an organic solvent (without CO ₂ ). Both options have the disadvantage of incomplete phospholipid extraction. Furthermore, both leave a solvent residue in the defatted egg powder, which results in protein denaturation. The neutral egg lipids obtained by supercritical CO ₂ extraction have negligible levels of highly unsaturated fatty acids, as shown in Example 3.

Arntfield et al. (JAOCS, 69, 1992, 823-825) show that egg proteins are substantially denatured after extraction with CO ₂ and methanol as a co-solvent. Using ethanol with supercritical CO ₂ results in incomplete extraction of phospholipids. Phosphatidylcholine is the most easily extracted phospholipid, but all other phospholipids have very low or no solubility and are not extracted (Teberliker et al., JAOCS, 78, 2002, 115-119). Schriener et al. (Journal of Food Lipids, 13, 2006, 36-56) shows that the majority of polyunsaturated fatty acids in egg yolk lipids are present in phosphatidylethanolamines that are not extracted by this method. .

  PCT Publication WO 02/092540 discloses the medical use of polar lipids containing HUFAs and blends of polar lipids with other oils. The extraction method is disclosed as using alcohol and centrifugation, but no further details are given. It is also disclosed that polar lipid-enriched fractions can be obtained as a by-product of edible seed oil extraction by an industrial method of degaming.

  A method for extracting HUFA-containing phospholipids from wet phospholipid-containing materials is disclosed in PCT Publication WO 2005/072477. Aliphatic alcohols, in particular isopropanol and / or n-propanol, are used. The substance containing the phospholipid is contacted with the water-soluble fatty alcohol at a temperature sufficiently high that the phospholipid dissolves in the solvent, during which time the denatured protein precipitates from the solution.

  Previously, DME was used to extract lipids from crude egg yolk (US Pat. No. 4,157,404) and dried egg powder (US Pat. No. 4,234,619). The method causes the lipid and protein components to be fractionated into separate streams. US Pat. No. 4,157,404 describes the extraction of lipids from crude egg yolk (water content 50-55%), but in this method the protein is denatured. The method also includes dehydrating the recovered lipid and water mixture to a moisture content of 20% or less, followed by phase separation of the neutral enriched phase and the complex lipid / water enriched phase. I need. US Pat. No. 4,234,619 discloses that proteins are not denatured when eggs are dry, in which case phospholipids can only be partially extracted. In the above method, phospholipids with a maximum yield of only 70% were obtained using whole egg powder that was spray-dried using DME within a temperature range of −30 ° C. to 40 ° C. The desired product of the present invention was an egg powder containing at least 30% its original phospholipid content and no cholesterol. A method for recovery and concentration of polyunsaturated fatty acids is not disclosed. Furthermore, due to the low extraction and separation temperatures used, it was not conceivable to separate the neutral lipids and complex lipids in the total lipid extract into separate fractions.

PCT publication WO 2004/066744 describes the extraction of lipids from an aqueous dairy stream using near-critical extraction when DME is the solvent. The publication further discloses that neither supercritical CO ₂ nor liquid DME can extract lipids in useful yields from dry howay protein concentrate (WPC) milk powder. The method does not disclose a method for extracting HUFA polar lipids from dried animal or plant tissue. Howay protein is not found in animal or plant tissues, and the resulting lipids do not contain polyunsaturated fatty acids.

  NZ535894 discloses the extraction of lipids from spray-dried dairy products containing milk fat globule membrane protein, a milk lipoprotein / lactose mixture resulting from the manufacture of skim milk powder. This protein is associated with the milk cream fraction and is not found in animal or plant tissues. Attempts to extract lipids from this milk powder stream with high lactose content (in this case high lactose content means at least 30% by weight of the total powder) by extraction with liquid DME have been unsuccessful, It was necessary to reduce the lactose content before production. Since the lipids do not contain HUFAs, there is no disclosure of a method for extracting HUFA lipids from dried animal or plant tissues. The residual powder after extraction still contains about 6-8% complex lipids.

  PCT Publication WO2006 / 058382 outlines a method for obtaining an extract from a range of substances using liquid DME. However, the extraction of HUFAs is not described, nor is the separation of complex lipids from neutral lipids described. The described method is a simple conventional method using liquid DME. Indeed, the only method described in some detail is to use liquid DME to obtain extracts from jojoba seeds that do not contain HUFAs.

  Determining whether the types of proteins and other complex carbohydrates present in products derived from animal and plant materials (and how to dry the materials) can successfully extract lipids is it is obvious. Proteins and complex carbohydrates present in plant or animal tissues are substantially different from those found in secondary products derived from animals, such as milk. Therefore, is it possible to extract lipids, particularly complex lipids containing polyunsaturated fatty acids, using dimethyl ether from plant or animal tissues containing proteins and complex carbohydrates in association with cells and tissues? It is generally impossible to predict whether or not with some certainty.

  Surprisingly, Applicants have found that liquid DME can be used as an effective extractant for obtaining HUFAs from plant or animal material, and in particular, residues in liquid extracts composed of neutral lipids and complex lipids. It has been discovered that DME allows the formation of a gummy phase containing complex lipids, which is then easily separated from neutral lipids.

It is an object of the present invention to provide a method for obtaining lipids containing polyunsaturated fatty acids, or at least to provide a useful alternative to other methods.
DESCRIPTION OF THE INVENTION In a first aspect, the present invention is a method for obtaining a lipid containing a highly unsaturated fatty acid from plant or animal material, comprising: (i) contacting the material with liquid dimethyl ether to contain the lipid. Providing a solution and a residue of plant or animal material; (ii) separating the solution from the residue of plant or animal material; and (iii) recovering lipid from the solution.

In certain preferred embodiments of the invention, the solution formed after contact with the material in step (i) contains neutral lipids and complex lipids.
Preferably, neutral lipids are recovered from the solution together with complex lipids. Thereafter, preferably, the neutral lipid is separated from the complex lipid.

  The complex lipid can form a gum phase with dissolved dimethyl ether during the recovery step (iii). Preferably, the gum phase containing complex lipids is separated from the solution containing neutral lipids.

  Preferably, the neutral lipid is separated from the complex lipid by phase separation. Centrifugation can also be used to aid separation. Heating can also be used prior to centrifugation. Next, the complex lipid is preferably dried by vacuum drying.

The method of the present invention preferably comprises contacting the lipid recovered from the solution in step (iii) with supercritical CO ₂ and the following step: (iv) the lipid recovered from the solution in step (iii) with supercritical CO ₂ And a step of obtaining a CO ₂ solution containing neutral lipid and a residue of complex lipid; (v) separating the CO ₂ solution containing neutral lipid from the residue of complex lipid; and (vi) The method further includes treating according to the step of recovering the neutral lipid from the CO ₂ solution.

In certain embodiments of the invention, the plant or animal material to be contacted with liquid dimethyl ether in step (i) is first treated with near-critical CO ₂ by the following steps: a. Contacting the material with near-critical CO ₂ to obtain a CO ₂ solution containing neutral lipids and a residue of plant or animal material; b. Separating the CO ₂ solution from the residue of plant or animal material; and c. The neutral lipid is treated according to the process of recovering from the CO ₂ solution.

  In a preferred method of the invention, the plant or animal material is dried or partially dried prior to use. Preferably, the plant or animal material is dried until the moisture in the material is less than 30% by weight, more preferably the moisture in the material is 5% by weight or more. Preferably, the plant or animal material is dried by freeze drying or by spray drying.

In certain embodiments of the invention, the plant or animal material is frozen wet biomass. Typically, frozen wet biomass is ground before extraction.
Preferably, one or more of the complex lipids are phospholipids, gangliosides, glycolipids, cerebrosides, or sphingolipids, typically phospholipids. The phospholipid includes any one or more of phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingomyelin, cardiolipin, plasmalogen, alkylacylphospholipid, phosphonolipid, lysophospholipid, ceramide aminoethylphosphonate, and phosphatidic acid. be able to. The glycolipids can include galactolipids, gangliolipids, sulphoquinovoysldiacylglycerides, tauroglycolipids, glycosphingophospholipids, and mannosyl lipids.

  The polyunsaturated fatty acid contained in the complex lipid is preferably, but not limited to, arachidonic acid (AA), α- and γ-linolenic acid, pinolenic acid, cyanidic acid, columbinic acid, dihomo Any one or more of linolenic acid, eicosatetraenoic acid, juniperonic acid, stearidonic acid, eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and docosahexaenoic acid (DHA) are included.

  In addition, the plant or animal material can be used as animal organs, animal glands, marine macro- and micro-algae, lipid-containing microorganisms (particularly filamentous fungi, algae, yeasts and bacteria) cultured by fermentation, shellfish, fish, It is also preferred to obtain from any one of the group consisting of marine invertebrates, eggs, plant seeds, plant leaves, plant needles, fern leaves, moss, and lichens.

In a preferred embodiment of the invention, the liquid dimethyl ether is near critical dimethyl ether.
In another aspect, the present invention provides a lipid containing a highly unsaturated fatty acid obtained by the method of the present invention.

In a further aspect, the present invention provides a complex lipid obtained by the method of the present invention.
In another aspect, the present invention provides a neutral lipid obtained by the method of the present invention.
In yet another aspect, the present invention provides plant or animal material from which lipids containing polyunsaturated fatty acids have been extracted by the method of the present invention.

  The present invention further provides the use of plant or animal material from which complex lipids containing polyunsaturated fatty acids have been extracted by the method of the present invention as nutraceutical, food supplements or as an enzyme source. To do.

DETAILED DESCRIPTION Definitions Fatty acid means any saturated or unsaturated aliphatic carboxylic acid typically having a hydrocarbon chain of 6 or more carbon atoms. The fatty acid has the number of carbon atoms (for example, C20), the number of unsaturated bond sites (for example, C20: 4), and the position of the first unsaturated bond site from the methyl terminal of the fatty acid (for example, C20: 4 w-3). And according to the number of carbons separating the unsaturated binding sites. Usually only if one carbon separates the unsaturated bond site (known as "methylene interrupted") and is conjugated (there is no carbon separating the unsaturated bond site) Abbreviated in short nomenclature, or when separated by more than one carbon (known as “non-methylene interrupted”), the methyl of the fatty acid The position of the carbon from the end is recorded (eg, 5, 11, 14 C20: 3). Fatty acids are a component of both neutral and complex lipids. In neutral lipids, only fatty acids bind to glycerol via ester bonds or ether bonds. Fatty acids can also exist in an unbound state, in which case they are referred to as “free fatty acids”. In complex lipids, fatty acids and other (polar) components bind to glycerol.

Polyunsaturated fatty acid (PUFA) means a fatty acid having two or more unsaturated binding sites.
Highly unsaturated fatty acid (HUFA) means a fatty acid having 3 or more unsaturated bonding sites and 18 or more carbon atoms in the fatty acid chain. Examples include arachidonic acid (AA), α- (ALA) and γ-linolenic acid (GLA), pinolenic acid, cyanidic acid, cholambic acid, dihomolinolenic acid, dihomopinolenic acid, juniperonic acid, stearidonic acid, eicosapentaenoic acid ( EPA), docosapentaenoic acid (DPA), and docosahexaenoic acid (DHA).

  Complex lipids included fatty acids (and closely related ethers, amines and hydrocarbon derivatives); polar phosphorus groups (usually phosphate esters or phosphates) and / or amino alcohols and / or carbohydrates; and glycerol , A lipid composed of at least three types of building blocks. Complex lipids include, but are not limited to, phospholipids, gangliosides, glycolipids, cerebrosides, and sphingophospholipids. Examples of phospholipids are phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylinositol (PI), sphingomyelin (SM), cardiolipin (CL), plasmalogen, lysophospholipid, and phosphatidic acid Is included.

  Neutral lipids are lipids that consist of one or two building blocks (both containing neither a polar phosphorus group nor a carbohydrate). The building blocks include fatty acids, glycerol, sterols, fatty alcohols, amines, carotenoids, and naturally occurring carbohydrates. Neutral lipids include, but are not limited to, fatty acids, mono-, di- and triacylglycerides, ceramides, N-acylethanolamines, sterols and sterol esters, carotenoids and carotenoid esters.

A DME hydrated complex lipid means a complex lipid that forms a weak bond with DME, similar to a lipid hydrated with water molecules.
The critical point means the point where the liquid state of the substance and the state of water vapor are the same.

  Supercritical means a pressure-temperature region exceeding the critical point of a substance. Beyond the critical point of a substance (but close to the critical point), the substance becomes a fluid state having both liquid and gaseous properties. The fluid has a density similar to a liquid and a viscosity and diffusivity similar to a gas.

  Sub-critical means a pressure-temperature region that is equal to or exceeds the vapor pressure of the material (but below the critical temperature). The terms “liquefied gas” and “compressed liquefied gas” can be used to denote the same region where the vapor pressure of the gas is at least 3 bar at the extraction temperature.

Near-critical means a pressure-temperature region close to the critical point of the material, and thus includes both subcritical and supercritical. Near-critical is the reduced temperature range 0.70 ≦ T _r ≦ 1.25 (where T _r is the temperature divided by the DME critical temperature T _c ); and the pressure range P> P for T <T _{c v} (in this case P _v is the vapor pressure) and the pressure range P> P _c (in this case P _c is the critical pressure) for T ≧ T _c .

  Nutraceutical means a product that has been isolated or purified from food, usually sold in a pharmaceutical form unrelated to food, has a physiological benefit and has been demonstrated to provide protection against chronic diseases.

The present invention is a method for obtaining a lipid containing a highly unsaturated fatty acid from a plant or animal material, wherein (i) the material is contacted with liquid dimethyl ether, a dimethyl ether solution containing the lipid, and a plant or animal. Obtaining a residue of material; (ii) separating the solution from the residue of plant or animal material; and (iii) recovering lipid from the solution.

  The plant or animal material can be any animal tissue or plant tissue containing lipids having HUFAs. In particular, the method comprises animal organs and glands, marine macro- and micro-algae, lipid-containing microorganisms cultured by fermentation, in particular filamentous fungi, algae, yeasts and bacteria; small marine animals (shellfish and invertebrates) , Eggs, and plant seeds. The plant or animal tissue may include plant or animal parts or whole materials, including cellular materials, proteins, lipids and carbohydrates, but secondary products derived from plants or animals (e.g., Does not include milk.

  DME is a gas at standard room temperature and pressure, but in liquid form is known to be an effective solvent for extracting substances from natural products. The liquid DME used in the method of the present invention is typically near critical DME. Preferably, the pressure of the liquid DME is at least equal to the vapor pressure at the temperature of extraction, more preferably at least 10 bar above the vapor pressure. The temperature is preferably in the range of 273 to 373K, more preferably in the range of 313 to 353K. The higher the extraction temperature, the higher the yield of complex lipids rich in highly unsaturated fatty acids. A typical extraction temperature is about 333K. A typical extraction pressure at this temperature is 40 bar, which is well above the vapor pressure of DME, ensuring maximum extraction of moisture when the biomass is wet.

  The lipids obtained by the method are generally a mixture of complex lipids with a range of bound HUFAs. The composition of the mixture will depend largely on the source of plant or animal material used. If the plant or animal material also contains neutral lipids, the neutral lipids will also be extracted in the method.

  Applicants have found that residual DME in a lipid extract composed of neutral lipids and complex lipids results in the formation of complex lipid-containing gum-like phase and neutral lipid-containing liquid phase (provided that the neutral lipid has a high concentration (5 (If not containing more than% by weight) free fatty acids and / or partial glycerides). The gum phase is a semi-solid liquid with a higher density than the neutral lipid-containing liquid phase. It is assumed that DME can form weak bonds with complex lipids (especially phospholipids) as well as the bonds formed between water and phospholipids. So-called DME-hydrated complex lipids in the gum phase can be easily separated from neutral lipids.

  The use of heat during the recovery of the extract, the ratio of neutral lipid to complex lipid in the lipid mixture, and the composition of the neutral lipid are important factors to promote the formation of DME hydrated complex lipids. is there. Recovery of the extract when the total lipid mixture contains about 50-90% neutral lipids without high levels of free fatty acids and / or partial glycosides and the lipid mixture is liquid at room temperature The process and subsequent degassing of DME from the extract by pressure loss and / or heating can lead to the formation of the complex. Separation of the gum and liquid phases is facilitated by the use of heat and / or centrifugation. The DME hydrated complex lipid phase thus obtained still contains some neutral lipids, but the neutral lipid phase has no complex lipids at all. This finding is particularly relevant to egg lipids and fish head lipids.

  Liquid DME can be used to extract both neutral and complex lipids from both wet and dry biomass to obtain a mixed extract after separation from DME. If the biomass is moist, moisture is also extracted and separated from the lipids by conventional means such as evaporation under vacuum, membrane separation, or in particular phase separation by centrifugation. Next, there is an option to further extract the mixed extract with near-critical carbon dioxide to separate and recover neutral lipids to obtain an extract richer in complex lipids containing HUFAs. The complex lipids are not hydrated and do not require further processing to remove water or DME.

  Plant or animal material can be extracted with near-critical carbon dioxide to extract neutral lipids prior to extraction with liquid DME. This sequence of processing steps also makes it possible to obtain extracts rich in complex lipids.

  Preferably, the pressure of the near critical carbon dioxide is at least 73.2 bar and the temperature is in the range of 304.2 to 373 K (supercritical region); or the pressure of the near critical carbon dioxide is above the vapor pressure The temperature is in the range of 273-304.1 K (subcritical region). More preferably, the carbon dioxide pressure is at least 250 bar and the temperature is in the range of 313-353K.

  An important element of certain embodiments of the present invention is the drying or partial drying of plant or animal material prior to extraction with liquid DME. Plant and animal materials typically have a moisture content of 60-80% by weight of the total material. Removing at least a portion of the moisture prior to extraction has the practical advantage that the yield of lipid is increased because the amount of moisture is reduced for a given amount of material. Therefore, the need for large capacity processing equipment is reduced or the throughput and lipid yield are increased for a fixed capacity processing plant. However, this method is also applicable to wet biomass, which avoids the cost of drying and reduces the lipids into the dry biomass matrix that can degrade lipids or prevent lipid extraction. It can be advantageous in that it inactivates enzymes that can cause encapsulation.

  Applicants have further discovered that, importantly, it is advantageous to dry the plant or animal material but not completely remove moisture. If the moisture content of the material to be extracted is reduced to a level of 30% by weight of the total material, the method of the present invention can be used without significantly degrading or denaturing enzymes and other proteins present in the material. It can be carried out. Therefore, the residues of plant or animal material after extraction can be used in various applications (eg, protein-rich and low-fat nutritional supplements such as body building products such as defatted cow liver) It may be particularly useful as a source of enzymes such as proteases, lipases, transglutaminases. The degradation of these enzymes is thought to limit the usefulness of the residue.

  Because complex lipids vary widely in their polarity, it is difficult to find a solvent or solvent mixture that can extract most of the phospholipids present in plant or animal tissues. Degreased residual material is used to extract residual lipids and / or complex non-lipid molecules during the extraction process so that non-lipid components such as enzymes can be extracted and used to be used as a nutraceutical. It is even more difficult to find a solvent system that does not. Surprisingly, if the material is dried prior to extraction using liquid DME used at a temperature of at least 40 ° C., all of the complex lipids and neutral lipids are not denatured without denaturing the residual defatted material. Applicants have found that high yields can be obtained.

General means The following four non-limiting general means show how the method of the invention can be carried out.

1. DME extraction a. The plant or animal tissue is dried to a moisture content of 30% or less. The moisture content is selected to ensure that the DME also contains water if necessary.
b. The plant material is ground to a particle size of 2 mm or less.
c. The plant or animal material is contacted with liquid DME at specified conditions.
d. Separate the loaded DME (laden DME) from the plant or animal material.
e. A HUFA enriched lipid extract is recovered from the DME.

If the lipid extract also contains neutral lipids, the following additional steps can also be performed:
f. The DME hydrated complex lipid is separated from the neutral lipid by phase separation.
g. Remove DME from the hydrated complex lipid.

In some cases, the residual defatted animal material can be further extracted with an aqueous solution to obtain the enzyme.
2. CO ₂ extraction after DME extraction a. The plant or animal tissue is dried to a moisture content of 10% or less.
b. The plant material is ground to a particle size of 2 mm or less.
c. The plant or animal material is contacted with liquid DME at specified conditions.
d. Separate the loaded DME from the plant or animal material.
e. Recover HUFA enriched complex and neutral lipid extract from the DME.
f. The HUFA-rich complex lipid extract is contacted with supercritical CO _2.
g. Neutral lipid-depleted HUFA-rich complex lipids are separated and recovered from supercritical CO ₂ and dissolved neutral lipids.
h. Recover the neutral lipid from the CO _2.

3. DME extraction after CO ₂ extraction a. The plant or animal tissue is dried to a moisture content of 10% or less.
b. The plant material is ground to a particle size of 2 mm or less.
c. The plant or animal material is contacted with supercritical CO ₂ at specified conditions.
d. Supercritical CO ₂ is separated from the neutral lipid depleted plant or animal material.
e. Recover the neutral lipid from the CO _2.
f. Plant or animal material is contacted with liquid DME at specified conditions.
g. Separate the loaded DME from the plant or animal material.
h. A HUFA enriched complex lipid extract is recovered from the DME.

4). DME extraction of wet biomass a. The biomass is frozen as necessary.
b. Frozen plant or animal material is ground to a particle size of 5 mm or less as needed.
c. The plant or animal material is contacted with liquid DME at specified conditions.
d. Separate the loaded DME from the plant or animal material.
e. Recover the HUFA-rich lipid extract and moisture from the DME.
f. Water is separated from the lipid.

If the lipid extract also contains neutral lipids, the following additional steps can also be performed:
g. HUFA enriched lipid material is contacted with supercritical CO ₂ at specified conditions.
h. The neutral-lipid depleted HUFA-rich complex lipid is separated and recovered from supercritical CO ₂ and dissolved neutral lipid.
i. Recovering the neutral lipids from the CO _2.

  In the above general means, spray-dried powder (for example, egg yolk powder) obtained from step a can be used so that step b is unnecessary in general means 1 to 3.

Example 1: Extraction of dried bovine liver About 8 kg of whole fresh bovine liver was obtained from a local meat processing plant. From the liver, cutaneous fat deposits, cartilage and skin were removed, and then the liver was cut into a plurality of large chunks. These chunks were passed through a mincing device to obtain a chunky paste. Next, 7913.5 g of minced liver was placed on lyophilized trays and these trays were then placed in the freezer until the solids were completely frozen. The tray was then placed in a lyophilizer and dried to a moisture content of about 2-5%. A solid yield of 31.9% was obtained, and 2526.7 g of material for trituration prior to extraction was obtained. The solid from the tray was ground in a knife mill equipped with a sieve plate having a hole size of ˜1 mm. The finely ground solid was then extracted with near-critical DME at 40 bar and 313K. Nearly critical DME 29.316 kg was continuously passed through the solid (2472.6 g) at a constant flow rate for 90 minutes. After passing the DME through the solid, it was continuously passed through a pressure reducing valve and a heat exchanger and put into a separation vessel, where the DME was immediately converted into a gas. Lipids precipitated from the gas and were recovered from the separation vessel. The DME was recycled to the extraction vessel via a condenser / subcooler heat exchanger and pump. 363.69 g of lipid was obtained in a yield of 13.96%. The lipid contains 53% phospholipids, of which 46.2% is phosphatidylcholine (PC), 10.2% is phosphatidylinositol (PI), and 2.3% is phosphatidylserine (PS), 16.6% is phosphatidylethanolamine (PE), 3.9% is sphingomyelin (SM), 6.6% is cardiolipin (CL), and 8% Not identified. Total lipid contains 4.5% arachidonic acid (AA), 7.4% docosapentaenoic acid (DPA), 2.1% eicosapentaenoic acid (EPA), and 5.9% α-linolenic acid (AA) did. The defatted liver can be used as a sports nutrition supplement.

Example 2: Extraction of bovine heart Approximately 8 kg of whole fresh bovine heart was obtained from a local meat processing plant. Skin fat deposits and cartilage were removed from the heart, and then the liver was cut into large chunks. These chunks were passed through the minting device. The minced heart was then placed on lyophilization trays and these trays were then placed in a freezer until the solids were completely frozen. The tray was then placed in a lyophilizer and dried to a moisture content of about 2-5%. A solid yield of 22.7% was obtained, giving 1725.7 g of material for trituration prior to extraction. The solid from the tray was ground in a knife mill equipped with a sieve plate having a hole size of ˜1 mm. The finely ground solid was then extracted with near-critical DME at 40 bar and 313K. 29.52 kg of near critical DME was continuously passed through the solid over a period of 90 minutes. After passing the DME through the solid, it was continuously passed through a pressure reducing valve and a heat exchanger and put into a separation vessel, where the DME was immediately converted into a gas. Lipids precipitated from the gas and were recovered from the separation vessel. The DME was recycled to the extraction vessel via a condenser / subcooler heat exchanger and pump. 202.71 g of lipid was obtained in a yield of 12.3%. The lipid contains 30.0% phospholipid, of which 28.3% is phosphatidylcholine (PC), 4.4% is phosphatidylinositol (PI) and 0% is phosphatidylserine ( PS), 13.7% is phosphatidylethanolamine (PE), 6.6% is sphingomyelin (SM), 27.9% is cardiolipin (CL), and 12.2% Was not identified. Total lipids contain 5.6% arachidonic acid (AA), 2.0% docosapentaenoic acid (DPA), 2.8% eicosapentaenoic acid (EPA), and 5.9% α-linolenic acid (AA) did.

Example 3: Extraction of spray-dried egg yolk with CO ₂ followed by DME This example first extracted neutral lipids from solid source material and then re-extracted with DME to enrich for HUFA It shows that complex lipid concentrates can be obtained. This example further shows that high extraction temperatures must be used to obtain high yields of complex lipids from spray-dried powders. 10.67 kg of spray-dried egg yolk powder was extracted with supercritical CO _{2 at} 300 bar and 313K. Egg oil containing only neutral lipids, with 530.34 kg of supercritical CO ₂ passed continuously through the solid and then through two vacuum stages, the pressure being first reduced to 90 bar at 313 K. (4.26 kg, yield 40.0 wt%) was recovered and then lowered to 58 bar at 323 K to recover a small amount of neutral lipid fraction (0.26 kg, yield 2.4 wt%). The neutral lipid contained less than 1% each of arachidonic acid and docosahexaenoic acid. Next, as in Examples 1 and 2, 2.98 kg of neutral lipid-depleted egg yolk powder was extracted with liquid DME 16.24 kg at 293 K and 40 bar for 60 minutes. 283.4 g of complex lipid extract containing no neutral lipid was obtained corresponding to a yield of 6.8% by mass of the whole fat egg yolk powder. The powder was re-extracted with 13.1 kg of liquid DME at 313 K as above for 50 minutes. An additional 191.3 g of complex lipid extract without neutral lipids was obtained, corresponding to an additional yield of 4.6% by weight of full fat egg yolk powder. Therefore, the total lipid yield was 53.8%. In order to obtain a high yield of complex lipid extract without neutral lipids, it is necessary to extract the powder at a temperature of at least 313K. This defatted egg yolk powder can be used for baking applications that require low fat.

Example 4: Extraction of spray-dried egg yolk with DME This example shows that a small amount of DME in the extract phase can be used to separate neutral lipids from complex lipids after DME extraction. 4.119 kg of spray-dried egg yolk powder was extracted with liquid DME at 323 K and 40 bar. 8.517 kg of near critical DME was continuously passed through the solid. After passing through the solid, the DME was continuously passed through a pressure reducing valve and a heat exchanger and placed in a heated separation vessel where the DME was immediately converted to gas. Lipids precipitated from the gas and were recovered from the separation vessel through a heating valve. The DME was recycled to the extraction vessel via a condenser / subcooler heat exchanger and pump. The liquid recovered from the separation vessel (2197.86 g, 53.3% yield) is heated to remove most of the residual DME, and then the liquid is centrifuged to remove the lipids from neutral lipids. Divided into chemical phase and DME hydrated complex lipid enriched phase. The neutral lipid enriched phase consists of 75.3% of total lipids and contains less than 1% by weight of complex lipids but contains neither arachidonic acid nor DHA. The polar lipid enriched phase consisted of 24.7% of the total lipid and contained more than 95% polar lipid. The polar lipid contained an arachidonic acid content of 5.89% and a DHA content of 2.46%.

Example 5 Extraction of Lyophilized Egg Yolk This example shows that freeze drying egg yolk improves lipid availability for extraction. Fresh eggs were purchased from a local store and then manually separated into egg yolk and egg white. The egg white was discarded. The egg yolk was mixed at room temperature and then added to a round bottom vacuum flask, frozen and then lyophilized. Next, 73.05 g of lyophilized egg yolk was extracted at 40 bar and 333 K with 598.1 g of liquid DME. 47.01 g of a yellow liquid extract containing 2% arachidonic acid and 1% DHA was obtained in a yield of 64% by weight, which is the same as the theoretical total lipid yield for egg yolk powder. The water solubility of residual egg yolk powder and non-extracted freeze-dried egg yolk powder was compared with spray-dried egg yolk powder (both defatted egg yolk powder and non-extracted egg yolk powder). Both fresh spray-dried egg yolk powder and extracted spray-dried egg yolk powder were insoluble in water, suggesting that the spray-drying process results in denaturation. Lyophilized protein (before and after extraction) was 22% soluble in water, while fresh egg yolk protein was 58% soluble. The extracted protein could be used as a low fat nutritional supplement.

Example 6: Extraction of lyophilized mussel powder using DME and CO ₂ This example shows that enzyme activity in defatted mussel solids is retained after lipid extraction. A frozen green-lipped mussel slurry is partially thawed and passed through a dejuicing apparatus to separate fine solids and liquid (slurry juice) from large chunks (solids). A portion of the slurry was set aside for processing as described in Example 12. The remaining slurry juice and solids were lyophilized separately and then first extracted with DME. The resulting crude extract was then re-extracted with supercritical CO _2. A DME extraction for comparison was also performed on the slurry (total powder in Table 1) that was directly frozen and then lyophilized. There was some variability in the yield from the abrasion and dehydration processes that produced some enzyme activity. Table 1 shows the lipid yield as a mass% of the dry powder, the extract complex lipid content, and the EPA and DHA content of the final product.

  Table 1: Extraction of HUFA-containing complex lipids from green mussels

  The phospholipid profile of the extract is as follows: phosphatidylcholine 31.9%, phosphatidylethanolamine 24.5%, phosphatidylinositol 3.9%, phosphatidylserine 3.1%, phosphonolipid 1.1%, ceramide 2-Aminoethylphosphonate 17.0%. The figures for phosphatidylcholine and phosphatidylethanolamine also include plasmalogens.

The phospholipase activity of the mussel powder after lipid removal was measured as follows: defatted green mussel powder (8 g) was mixed with 40 ml of distilled water and then centrifuged. An aliquot (20 ml) of the supernatant mussel preparation was added to 1 g of a phospholipid mixture model containing PC-24%, PE 34% and PS 12%, emulsified at 40 ° C. and then maintained at this temperature for 16 hours. A sample (0.2 ml) of the reaction mixture was analyzed for phospholipid composition by ³¹ P-NMR. The degree of hydrolysis of phospholipids is shown in Table 2. In Table 2, L means lyso- (one fatty acid hydrolyzed from parent phospholipid) and G is glycero- (hydrolyzed from parent phospholipid). Both fatty acids) and tot means total intact and hydrolyzed phospholipids. A large degree of hydrolysis was clearly observed for PC and PE. However, the major class of total hydrolysis plus the complete phospholipid profile was significantly different from that of the starting material, suggesting that other reactions were performed. ^{The 31} P NMR spectrum formed several new unidentified peaks that could suggest a product with phospholipase C activity, which could explain this discrepancy.

  Table 2: Phospholipid hydrolysis using mussel enzyme extract

Example 7: Extraction of Hoki head using DME This example is based on the ocean because a temporary complex is formed between DME and phospholipid after DME extraction (DME-hydration) (Marine-based) shows that neutral lipids can be separated from complex lipids. The frozen hoki fish head was passed through a mincer. The minced heads were then placed on a lyophilizer tray, frozen, and then lyophilized. The dried head mince was then further ground with a knife mill to a powder and extracted with DME at 40 bar and 333K. Using the general method described in Example 4, 1970.6 g of powder was extracted with 15.408 kg of DME. A brown liquid lipid enriched extract was obtained, which upon standing, began to separate into a neutral lipid enriched phase and a phospholipid enriched phase. The extract was centrifuged to facilitate phase separation. The upper neutral lipid phase contained only 2.5% phospholipids. The lower “gum” phase contained 19.2% by weight of DME-hydrated phospholipid. Next, DME in the lower phase was removed under vacuum to obtain an extract containing 33.2% phospholipid and 0.5% ganglioside. This complex lipid concentrate contained 5.8% EPA, 12.7% DHA and 3.6% other HUFA.

Example 8: Extraction of lemon fish meat with DME This example shows that complex lipids very rich in HUFA can be extracted from fish meat. Fresh lemon fish meat was cut into multiple cubes and then lyophilized. The dried fish cubes were then further ground with a knife mill to a powder and extracted with DME at 40 bar and 333K. Using the general method described in Examples 1 and 2, 135.95 g of powder was extracted with 886.7 g of DME. A phospholipid-rich (64% by weight) yellow semi-solid extract was obtained in a yield of 2.6%. The phospholipid fraction of the extract contained PC 46.2%, PI 7.9%, PS 3.5%, PE 25.0%, SM 5.2% and CL 7.9%. The extract was particularly rich in DHA, which was 24.9% of total fatty acids. The complex lipid extract also contained 4.5% DPA, 5.3% EPA and 6.7% AA. Non-denatured fish protein can be used as a food supplement.

Example 9: extraction of sheep and porcine pancreas by DME, reextracted this embodiment of lipid extract with supercritical CO _2, using supercritical CO _2, reextracted neutral lipids from the crude DME extracts And that both the active phospholipase enzyme and the proteolytic enzyme can be extracted from the residual solid. Lyophilized bovine and porcine pancreas samples were extracted at 40 bar and 333 K using liquid DME. Using the general method described in Examples 1 and 2, 120.72 g of bovine pancreas was extracted with DME 1193.4 g. A yellow / green semi-solid extract very rich in neutral lipids was obtained in a yield of 44.8%. The extract contained only 19% phospholipids, 0.7% AA and 0.7% DPA. Using the general method described in Examples 1 and 2, 120.18 g of porcine pancreas was extracted with 1240.2 g of DME. A yellow semi-solid extract very rich in neutral lipids was obtained in 24.0% yield. The extract contained only 13% phospholipid, AA 1.5%, but contained neither EPA nor DPA. The bovine and porcine crude pancreatic extracts were then re-extracted with supercritical CO _{2 at} 300 bar and 333 K until neutral lipids were no longer recovered as extracts. Thereafter, the extract and residual complex lipid concentrate were reanalyzed. The bovine complex lipid contained 2.3% AA, 1.4% EPA and 1.8% ALA. The porcine complex lipid contained 4.8% AA and less than 1% each of EPA and DPA. Sheep and porcine residual defatted pancreatic solids were then tested for their proteolytic and phospholipase activities.

Phospholipase activity was measured as follows: defatted bovine pancreas (0.65 g) and defatted porcine pancreas (0.98 g) solids were mixed in distilled water (20 ml) and then centrifuged. An aliquot (2 ml) of the supernatant pancreas preparation was added to an emulsion of a phospholipid mixture model (1 g) containing PC-24%, PE 34% and PS 12% in water (10 ml) and maintained at 40 ° C. for 16 hours. . A sample (0.2 ml) of the reaction mixture was analyzed for phospholipid composition by ³¹ P-NMR. The degree of hydrolysis of phospholipids is shown in Table 3. In Table 3, L means lyso- (one fatty acid hydrolyzed from parent phospholipid) and G is glycero- (hydrolyzed from parent phospholipid). Both fatty acids). The porcine pancreas showed significant phospholipase A2 activity, preferential hydrolysis of PE>PS> PC. The bovine pancreas showed very low phospholipase A2 activity against PE and PC, but similar levels of hydrolysis of PS compared to porcine pancreas.

  Table 3: Phospholipase A2 activity

Protease activity remaining after DME extraction was measured as follows. DME-extracted porcine pancreas freeze-dried powder (0.9834 g) was extracted with 25 ml of 100 mM CaCl ₂ (39 mg / ml), and DME-extracted bovine pancreas (0.65 g) was extracted with 25 ml of 100 mM CaCl ₂ (26 mg / ml). . The extract was tested before and after enzyme self-activation and compared to a standard pancreas extract prepared from frozen porcine and sheep pancreas. Yields are shown in Table 4 for DME extracted powder and in Table 5 for frozen reference samples.

  Table 4: Pancreatic protease yield from DME extracted freeze-dried pancreas powder

  Table 5: Standard protease levels extracted and activated from frozen pigs and sheep pancreas

  Data are based on extraction of 25 g sheep and porcine pancreas using standard conditions. Enzyme activity was measured after activation was complete, which was determined by a slight decrease in trypsin activity after the maximum level of trypsin activity was reached. The substrate SAAPLpNA used to detect elastase II activity is also hydrolyzed by elastase I and chymotrypsin. Therefore, elastase II activity was estimated using this substrate.

  Extraction of lyophilized pancreas with DME hardly reduces the total protease content of porcine pancreas. The low level of enzyme activity detected in lyophilized bovine pancreas previously extracted by DME can be attributed to species variation and / or age of the animal from which the pancreas was collected. A significant level of self-activation was observed in the DME-treated powder, as judged by the first detection of relatively high protease levels prior to enzyme precursor activation induced by pH adjustment. The initial trypsin activity detected was sufficient for complete activation of the enzyme precursor when the extract pH was adjusted to a value more suitable for activation (eg, pH 8.5). In contrast, activation of pancreatic enzyme precursors from frozen pancreas requires the addition of exogenous trypsin.

  Comparison of proteolytic profiles obtained from activated frozen pancreas and lyophilized pancreas extracted from DME shows that considerable enzymatic activity is retained after DME extraction. The typical extraction efficiency of elastase I from frozen pancreas resulted in a yield of 0.67 μmol / min / g tissue, whereas that of elastase I obtained from DME-extracted lyophilized porcine pancreas was 67.4 μmol / min. A yield of min / g tissue was produced. Trypsin yields appear to be lower than expected, but this explains the higher yields than expected for other proteases that undergo trypsin-mediated hydrolysis (decreasing their activity during extraction from frozen pancreas) be able to.

Example 10 Extraction of Hoki Liver with DME This example shows that lipids containing highly unsaturated fatty acids can be extracted directly from wet biomass. Commercially obtained frozen whole hoki fish liver was passed through a Urschel grinder with a large overall size, and the liver was cut into multiple chunks. The macerated liver was then extracted with DME for 2 hours at 60 ° C. and 40 bar. 31.996 kg of DME was passed through 6.7427 kg of wet liver. 2.234 kg of an extract composed of water and lipids containing highly unsaturated fatty acids was obtained. The partially extracted residual solid was then re-mixed and re-extracted with DME for 3 hours at the same conditions. 48.46 kg of DME was passed through the liver to recover an additional 1.834 kg of extract, which was mostly water. A total of 2.3082 kg of oil was obtained after water evaporation. This oil contained 9.35% DHA, 1.43% DPA, 4.91% EPA, 1.3% C20: 4w-3, 0.6% AA and C18: 3 and C18: 4w-3 1.9% . Residual solids were tested for trans-glutaminase activity, which was inactivated by this extraction process.

Example 11: Extraction of pine tree seeds with DME to obtain lipids rich in non-methylene interrupted fatty acids This example involves extracting pine seeds with DME to obtain a lipid extract rich in non-methylene interrupted fatty acids. Show what you can do. Commercially available seeds of the pine species Biota Orientalis were partially cold-pressed before extraction with DME. The remaining cold-pressed seed cake contained about 35% by weight neutral oil (26% on a prepressed basis). The pressed seed cake was extracted with DME for 150 minutes at 60 ° C. and 40 bar. 37.06 kg of DME was passed through 14.0385 kg of partially compressed seed. 5.942 kg of extract was obtained, which was a mixture of neutral lipids, complex lipids and water. The extract was separated into phases by centrifugation. 4.847 kg of neutral lipid oil was isolated as the upper phase. This oil contained 9.9% juniperonic acid (C20: 4 non-methylene interrupted fatty acid), 4.3% cyanidic acid (C20: 3 non-methylene interrupted fatty acid) and 33.2% α-linolenic acid. 0.488 g of unidentified complex lipid was isolated as the mesophase. This had a fatty acid composition similar to the major lipid product.

Example 12 Extraction of Green Mussel Slurry with DME, followed by Separation of Neutral and Complex Lipids by Supercritical CO ₂ Extraction This example extracts lipids containing highly unsaturated fatty acids from animal tissue slurries Show what you can do. The slurry of green mussel solid produced in Example 6 was extracted without drying. In this case, a slurry containing finely divided mussel solids is fed into the extraction vessel at high pressure and simultaneously at a pressure of 40 bar and an extraction temperature of 60 ° C. in a vertical static mixer inside the vessel. Contacted with DME. The extracted solid was deposited on the bottom of the extraction vessel. DME and dissolved lipids and water were removed from the top of the vessel and passed through a vacuum valve and heat exchanger leading to a separation vessel as described in the previous examples. Enriched with HUFA (C18: 3 and C18: 4w-3 3.4% after removal of moisture under vacuum by contacting 6.1.59 kg of mussel slurry solution at 333 K and 40 bar with DME 52.906 kg; EPA 18.7 %, DHA 11.1%) 80.4 g of extract containing complex lipids and neutral lipids were obtained. The residual solid was lyophilized and the lipid yield was measured on a dry basis and found to be 9.0% by weight. The lyophilized solid was then triturated and re-extracted by the same method as Example 6, but only a further yield of 0.3% by weight was obtained, from the slurry Indicates that the extraction is almost complete. Next, 49.74 g of lipid extract was re-extracted with supercritical CO _{2 at} 333 K and 300 bar to give 29.10 g of neutral lipid (58.4% yield based on total lipid). The extract was rich in HUFA (C18: 3 and C18: 4w-3 4.0%; EPA 20.2%, DHA 11.0%). Residues that were almost all of the type of complex lipids described in Example 6 were also rich in HUFA (C18: 3 and C18: 4w-3 2.4%; EPA 17.2%, DHA 12.6%).

Example 13: extraction of the lyophilized krill with supercritical CO ₂ and subsequent DME, with supercritical CO ₂ re-extraction following the DME extraction, separation This example neutral lipids from complex lipids rich in HUFA By extracting lipids containing polyunsaturated fatty acids from lyophilized krill, first by extracting neutral lipids by extraction with CO ₂ and then by extraction with DME, by extracting complex lipids rich in HUFA, or It is shown that total lipids can be extracted from krill using DME and then extracted by re-extracting the total lipid extract with supercritical CO ₂ to remove neutral lipids. 180.12 g of lyophilized krill powder containing 12.2% lipid was extracted with supercritical CO _{2 at} 300 bar and 314 K to give 11.28 g of lipid. The residual krill powder was then extracted with DME at 40 bar and 332K to give 3.30 g of phospholipid-rich lipid containing 20% EPA, 15.6% DHA and 38% total HUFA. 3.0603 kg of 2nd krill powder containing 21.4% lipid was extracted on a pilot scale with 17.271 kg DME at 40 bar and 357 K to give 652.1 g lipid-enriched extract, which was present Among the total fatty acids, EPA contained 14.0% and DHA 9.0%. Next, 100.32 g of this lipid enriched extract was re-extracted with 26.21 kg of supercritical CO _{2 at} 300 bar and 314 K to be very rich in phospholipids (76.6%) (EPA 28.8%). 33.04 g of non-extracted lipid residue (containing 21.9% DHA and 55.6% total HUFA).

Example 14 Extraction of Lipids from Wet and Dry Mortierella alpina Biomass Using DME In this example, the microorganism Mortierella alpina (strain IRL176) is fermented to yield arachidonic acid rich lipids. Obtained. The biomass was then extracted either as wet biomass or as a dry material to obtain an extract that was very rich in arachidonic acid. A 150 ml roux flask was made with 25 ml of potato dextrose agar (PDA). The roux flask was inoculated with 0.1 ml of spore stock and incubated at room temperature for 1 month. The concentrated spore stock was scraped from the surface of the PDA roux flask and inoculated into a 500 ml baffled shake flask with 200 ml of potato dextrose medium. The seed shake flask was incubated for 96 hours at 25 ° C. on a rotary shaker (180 rpm). A 15 ml sample of the seed shake flask was inoculated into a 13 × 500 ml (2000 ml total) unbaffled shake flask containing 40 g / L glucose and 10 g / L yeast. Production shake flasks were incubated for 7 days at 25 ° C. on a rotary shaker (180 rpm). The culture was harvested and the biomass was collected by filtration (filter paper No. 1). The collected cells were washed with 60 ° C. water at a concentration of 1: 1 (vol / vol). The dried cell weight was 12.2 g / L. Although 218.85 g of fresh wet biomass was extracted by DME, only 1.89 g of extract with low levels of lipids was recovered. This suggested that the cells were not ruptured. Next, 150.14 g of a second batch of fresh wet biomass was frozen and subsequently ground before being extracted with 2.416 kg of DME at 333 K and 40 bar. Freezing and subsequent attrition ruptured the cells and allowed oil extraction. A mixture of oil (6.51 g) and water (99.53 g) was extracted. The oil contained 31.8% arachidonic acid, 13.8% GLA and 55.9% total PUFA. Next, the residual biomass after extraction (37.34 g) was dried overnight at 313 K in a forced convection oven to give a final dry mass of 30.02 g. The dried biomass was then ground with a mortar and pestle. Next, 27.61 g of this dried biomass was extracted with 0.840 kg of DME at 333 K and 40 bar to recover an additional 3.87 g of lipid, which was arachidonic acid 33.2%, GLA 14.1% and total PUFA 57.3. %.

Example 15: Extraction of highly unsaturated algal lipids using DME 58.29 g of wet biomass obtained from mixed and organic nutrient fermentation of Nitzschia Laevis was frozen and then Extracted at 40 bar and 333K. 53.10 g of total extract was obtained, consisting of 43.55 g of water and 9.55 g of neutral and complex lipids (containing 2.2% AA, 11.8% EPA and 2.8% DHA). Met. The lipid mixture was separated from the water by evaporation under vacuum. 2.161 g of dry lipid mixture is extracted at 108 bar and 333 K with 108 g of supercritical CO ₂ to give 0.560 g of neutral lipids containing only AA 1.4%, EPA 8.2% and DHA 2.2%. Obtained. The residual complex lipid material after extraction contained AA 4.2%, EPA 20.0% and DHA 3.4%.

INDUSTRIAL APPLICABILITY The method of the present invention can be used, for example, for dry or partially dried plants or seeds (including marine or terrestrial species) or animal articles (including marine or terrestrial species or microorganisms). It is useful for extracting highly unsaturated lipids (fatty acids) from such substances. Polyunsaturated lipids are critical for infant brain and vision development and may also be beneficial for cardiovascular health, mental health, immune and inflammatory conditions.

Claims (32)

A method for obtaining lipids containing polyunsaturated fatty acids from plant or animal materials,
(I) contacting the material with liquid dimethyl ether to obtain a dimethyl ether solution containing lipid and a residue of plant or animal material;
(Ii) separating the solution from the residue of plant or animal material; and (iii) recovering lipid from the solution.   The method of claim 1, wherein the solution formed after contact with the material in step (i) contains neutral lipids and complex lipids.   The method of claim 2, wherein the neutral lipid is recovered from the solution together with the complex lipid.   4. The method of claim 3, wherein the neutral lipid is separated from the complex lipid.   The process of claim 2 wherein the complex lipid forms a gum phase with dissolved dimethyl ether during the recovery step (iii).   6. The method of claim 5, wherein the gum phase containing complex lipids is separated from the solution containing neutral lipids.   The method of claim 6, wherein the neutral lipid is separated from the complex lipid by phase separation.   The method of claim 6, wherein the neutral lipid is separated from the complex lipid by centrifugation.   9. The method of claim 8, wherein heating is used prior to centrifugation.   The method according to any one of claims 8 to 10, wherein the complex lipid is dried by vacuum drying. The lipid recovered from the solution in step (iii) is converted by near-critical CO ₂ into the following steps:
(Iv) contacting the lipid recovered from the solution in step (iii) with near-critical CO ₂ to obtain a CO ₂ solution containing neutral lipid and a complex lipid residue;
Further comprising: (v) separating the CO ₂ solution containing neutral lipids from the residue of complex lipids; and (vi) recovering the neutral lipids from the CO ₂ solution. Item 11. The method according to any one of Items 1 to 10. The plant or animal material to be contacted with liquid dimethyl ether in step (i) is first subjected to the following steps by near-critical CO ₂ :
a. Contacting the material with near-critical CO ₂ to obtain a CO ₂ solution containing neutral lipids and a residue of plant or animal material;
b. Separating the CO ₂ solution from the residue of plant or animal material; and c. The method according to claim 1, wherein the treatment is performed according to a step of recovering neutral lipid from the CO ₂ solution.   13. A method according to any of claims 1 to 12, wherein the plant or animal material is dried or partially dried prior to use.   14. The method of claim 13, wherein the plant or animal material is dried until the moisture in the material is less than 30% by weight.   15. A method according to claim 14, wherein the plant or animal material is dried such that the moisture in the material is 5% by weight or more.   16. A method according to any of claims 13 to 15, wherein the plant or animal material is dried by freeze drying or by spray drying.   12. A method according to any of claims 1 to 11, wherein the plant or animal material is frozen wet biomass.   The method of claim 17, wherein the frozen wet biomass is ground prior to step (i).   The method according to claim 1, wherein the one or more complex lipids are phospholipids, gangliosides, glycolipids, cerebrosides, or sphingolipids.   The method according to any one of claims 1 to 19, wherein the one or more complex lipids are phospholipids.   The phospholipid contains at least one of phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingomyelin, cardiolipin, plasmalogen, alkylacyl phospholipid, phosphonolipid, lysophospholipid, ceramide aminoethylphosphonate, and phosphatidic acid. 21. The method of claim 20, comprising.   20. The method of claim 19, wherein the glycolipid comprises galactolipid, ganglioside, sulfoquinovoice diacylglyceride, tauroglycolipid, glycosphingophospholipid, and mannosyl lipid.   Polyunsaturated fatty acids contained in the lipid are, but not limited to, arachidonic acid, α- and γ-linolenic acid, pinolenic acid, cyanidic acid, cholambic acid, dihomolinolenic acid, eicosatetraenoic acid, juniperonic acid The method according to any one of claims 1 to 22, comprising any one or more of:, stearidonic acid, eicosapentaenoic acid, docosapentaenoic acid, and docosahexaenoic acid.   The plant or animal material is an animal organ, animal gland, marine macro- and micro-algae, filamentous fungi, bacteria, yeast, shellfish, fish, marine invertebrates, eggs, plant seeds, plant leaves, plants 24. The method according to any one of claims 1 to 23, obtained from any one of the group consisting of: needles, fern leaves, moss, and lichens.   25. A process according to any of claims 1 to 24, wherein the liquid dimethyl ether is near critical dimethyl ether.   The lipid containing the highly unsaturated fatty acid obtained by the method in any one of Claims 1-25.   The complex lipid obtained by the method in any one of Claims 1-25.   The neutral lipid obtained by the method in any one of Claims 1-25.   A plant or animal material from which a highly unsaturated fatty acid-containing lipid is extracted by the method according to any one of claims 1 to 25.   Use of the plant or animal material according to claim 29 as a nutraceutical.   30. Use of the plant or animal material according to claim 29 as a food supplement.   30. Use of the plant or animal material according to claim 29 as an enzyme feedstock.